This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

所有小鼠饲养，并按照指南的护理和使用美国国立卫生研究院实验动物的处理，并参加了一个NHLBI动物护理和使用委员会批准的协议。女B6.SJL-PTPRC（D）Pep3（B）/ BoyJ（B6.SJL）和C57BL / 6小鼠，6〜12周龄，分别作为供体和受体，分别为。 在表1中提供的材料，试剂和设备的列表。 鼠标HSPCs 1。乐高传导和骨髓移植在执行经过认证的生物安全2级（BSL-2）橱柜（组织培养罩）下面介绍的程序。 <ol><li>生产LEGO向量编码5FPS的<ol><li>使用慢病毒“基因本体论”（LEGO）的载体质粒，分别来自强组成SFFV启动，包括乐高，Cer2（蔚蓝），LEGO-G2（EGFP），LEGO-V2（金星），LEGO-T2的编码不同的FP （TdTomato），以及LEGO-C2（mCherry）鲍里斯Fehse博士（大学医学中心汉堡Eppendorf公司，德国汉堡）友情提供;现在也可以从Addgene 5-7。 </li><li>以产生LEGO矢量，种子5×10 6个293T细胞以10 cm的培养皿（使用12个培养皿的每个向量）24小时在转染前在8ml I10介质（IMDM补充有10％FCS和谷氨酰胺/青霉素/链霉素）中。 </li><li>转用稍微修改CAPHOS包建议的细胞磷酸钙： <ol><li>对于每块板组合15微克LEGO载体，12微克pCDNA3.HIVgag / pol.4xCTE，4微克上一页达，和1微克pMD2.G-VSV-G质粒中存在的50微升2.5M的氯化钙2，加水至产生500微升总体积。 </li><li>沉淀的DNA的质粒用500μl2×HEBS（HEPES缓冲盐水）中，并添加到293T细胞中的各板前孵育20分钟，在室温下进行。 </li><li>孵育细胞6小时，在37℃的5％CO 2培养箱;删除媒体和补充新鲜培养基6小时后转染。 </li></ol></li><li>收集培养物上清液含有慢病毒颗粒从各板，每天，持续3天。每一天，下面收集上清液加入新鲜I10媒体每块板。 <ol><li>通过0.22微米的微孔，游泳池过滤并在71,900 XG和4℃下浓缩上清液通过超速离心上的SW28转子2小时。 </li><li>悬浮上清液1毫升StemSpan媒体。分装慢股200微升，并储存在-80℃。 </li></ol></li></ol></li><li>慢病毒颗粒的滴定 通过利用转导NIH3T3细胞单位/毫升的定量测定LEGO载体颗粒的收集上清液的效价。滴度允许需要导致感染复数（MOI）的所需的相对多重每个矢量的上清液用于实验对象的体积的计算细胞。 <ol><li>板5×10 4个NIH3T3细胞在2ml I10媒体的前一天滴定的每孔（12孔板）的细胞。 </li><li>制备载体上清液的5倍连续稀释液在添加有硫酸鱼精蛋白I10媒体（4毫克/毫升为1000倍）。 </li><li>从细胞中取出介质，并更换500微升含2，0.8，0.32和0.128微升浓病毒相当于上清液稀释。 </li><li>确定细胞每孔存在于转导的时间数（否），计算一个控制良好。 </li><li>转导后6小时后，加入2mL I10媒体的孵育3天，在37℃，5％CO 2。 </li><li>从用胰蛋白酶-EDTA的每个孔收获细胞，并测定荧光细胞（％FP）经由流的百分比术。 </li><li>计算滴度如下，平均每孔（稀释）得到的滴度产生介于1和50％的％FP：效价（载体颗粒/毫升）=否矢量上清液（毫升）的x％的FP /音量。平均得到的各稀释液，如果％FP是介于1和50％的效价。 </li></ol></li><li>小鼠细胞收集，净化，转导，并移植（ 图1A示意性地显示从马利德等 4改编）。 <ol><li>经批准的程序牺牲供体小鼠。收获骨髓前后肢：解剖肱骨，股骨和胫骨，彻底去除所有肌肉，韧带和多余的组织从骨骼，贯穿各骨尽可能接近到接头的端部，并冲洗骨髓出用27-0 16G针含I10媒体注射器的骨头。 </li><li>沉淀全骨髓细胞10分钟，在500 XG，4℃，裂解红细胞两次用50ml ACK缓冲液中。将细胞再悬浮于10ml I10媒体。 </li><li>根据修改后的试剂盒protoco净化谱系阴性（Lin-的）祖细胞的MACS谱系耗尽试剂盒L： <ol><li>使用30微升抗生物素微珠代替10微升/ 10 7个细胞;完成清洗步骤与I2介质（IMDM补充有2％FCS）代替缓冲液。 </li><li>将培养与生物素标记抗体鸡尾酒和抗生物素微珠保温之间的一个洗涤步骤;和平衡色谱柱与I10，而不是缓冲区。 </li></ol></li><li>培养Lin-的细胞48小时，以5×10 5细胞/ ml，在StemSpan媒体的起始密度补充有10纳克/毫升鼠IL-3，100毫微克/毫升鼠IL-11，100纳克/毫升人FLT-3配位体，和50毫微克/毫升小鼠干细胞因子。 </li><li>转让1至4×10 5个细胞于12孔板中包被Retronectin和转导与LEGO上清，每在感染复数为6-7（MOI），以实现对每个FP 50％的表达效率，在StemSpan的存在介质和细胞因子的上面详述和4微克/毫升的硫酸鱼精蛋白。 </li><li>转导细胞每个LEGO分别为矢量描述的步骤1.3.5及以下转导的细胞混合（称为混合）或同时共转导细胞与所有5 LEGO载体上清液（称为钴）。 24小时后取出电池，用StemSpan媒体洗一次无细胞因子和它们悬浮在StemSpan媒体无细胞因子进行移植，或共聚焦显微镜前，将其转移至新鲜培养基与细胞因子和培养更多的96小时和流式细胞仪。 <ol><li>通过注入导Lin-的HSPCs经尾静脉注射入11 Gy的单剂量预照射的收件人（前6-18小时，从而移植）的收件人进行骨髓移植，在100μl的体积，在每只小鼠的细胞的剂量对应于1 -4×10 5个Lin-的细胞最初镀转导。在每个实验中，移植动物的整个队列与转导的供体骨髓细胞的相同的人口。 </li><li>对于转导ħ 体外表征的SPC，培养的细胞在StemSpan额外4-5天补充有细胞因子和通过流式细胞仪对转染效率进行分析，并通过显微镜进行颜色的多样性。 <ol><li>牺牲个别小鼠和检索的组织和器官的成像，在不同的时间点达120天移植后。 <ol><li>用乙醇喷雾鼠标的皮毛和切割的腹侧避免头发散发肌肤。 </li><li>根据标准程序，脾，腘淋巴结，肺，心脏，皮肤，脂肪组织，骨骼肌，肾脏，肝脏，以及头部，以获得颅盖骨解剖和切除。 </li><li>另外，使用颅骨或建立骨窗实时成像重复随着时间的推移2,8,9或图象表面的器官，如在活的动物的皮肤和淋巴结。 </li></ol></li><li>将各个器官的35毫米数0玻璃盖培养皿中50-10056，L PBS成像它们完好无如步骤2.5中所述切片，固定或进一步加工。 </li><li>对于容积成像，保持器官的冷PBS备用。对于时间推移成像立即着手，含20毫米的HEPES 10％胎牛血清，37℃成像器官的DMEM </li></ol></li></ol></li></ol></li></ol> 2，激光共聚焦和双光子显微镜和图像分析<ol><li>执行使用装有多线氩，二极管561纳米，氦氖594纳米，氦氖633纳米可见激光徕卡SP5的五个通道共聚焦和多系统显微成像。使用双光子模式下，脉冲飞秒钛：蓝宝石（TI-SA）激光器，可调谐的激励，从680到1,080 nm处色散校正。使用在涉及红移的FP（tdTomato和mCherry），光学参量振荡器（OPO）激光器的实验来尽量延长输出波长范围为1,030-1,300纳米，依次或同时用叔他的Ti-萨激光。 </li><li>使用HCX-IRAPO-L 25X / 0.95 NA水浸渍目标（WD = 2.5 mm）表示时HC-PLAPO-CS 20X / 0.70 NA干物镜（WD = 0.6 MM），或HC-PL-IRAPO 40X图像的各种组织/1.1 NA水浸物镜（WD = 0.6毫米）。 </li><li>得到收集发射光的λ栈（xyλ）共焦荧光光谱中为5nm-带宽从BM细胞转导的单个FP LEGO矢量以及控制的可见光谱中的，未移植BM。 <ol><li>图像处理与Leica LAS-AF V2.4.1软件创建的所有5张参考光谱，以及自发荧光（ 图2A）。使用个别的FP光谱和荧光亮度的差（表示为％的EGFP）大仓（79％），金星（156％），tdTomato（283％）和mCherry（47％）的设定5序列成像通道，如下所示：蔚蓝（458 / 468-482纳米），绿色荧光蛋白（488 / 496-514纳米），金星（514 / 523-558纳米），tdTomato（561 / 579-597纳米）和mCherry（594 / 618-670纳米） （ <str翁&gt;图2B）。 </li><li>验证准确的FP分离，如转导的细胞与每个FP仅在相应的信道可见和不存在来自其他地方（ 图2C）。另外，该定义的多通道方法创造了一个独特的5频道“COLORPRINT”，使后续的颜色分析和克隆跟踪，并具有减少采集时间相比，光谱成像的优点。 </li></ol></li><li>单独的荧光发射，在2光子模式中，通过高效率的自定义分色镜和收集具有四个通道的非退扫描检测器（NDD）如下：二向色镜在568 nm的后跟一个分色镜在465nm处，接着à四十五分之四百五十二nm发射滤波器，倍频或自发荧光和蔚蓝，对于绿色荧光蛋白或金星在tdTomato或mCherryà四十○分之五百二十五nm发射滤波器和648 nm的分色镜和六十零分之六百二nm发射滤波器。 <ol><li>通过双光子显微镜我揭示的结构信息ntrinsic对比成像（如上文所述）的组织自发荧光的由弹性蛋白，NADH，（激发在780nm处）和二次谐波产生的从骨和纤维状胶原信号（SHG）（激发在收集在背散射920纳米或860纳米方向）10,11。 </li></ol></li><li>对于3D体绘制，沿z轴收集一系列的xyz的图像（通常为1×1×4微米的3体素尺寸）在5微米的间隔的范围内的深度（150-300微米）在整个骨髓和其他组织中，在使用软件的瓦片函数大的区域，以自动生成缝合卷包含整个胸骨窝，约2.5×1.2 平方毫米（XY）和300微米（Z）（ 图2D-2E）及电影1。 <ol><li>颅骨图像直接通过颅骨骨髓切片没有2,8。 </li><li>沿矢状平面（平分），然后部分胸骨在横向通过切割面3,4,12 2胸骨福斯和图像之间的联合（概述图1B改编自高久等[3]）。 </li></ol></li><li>四维时间推移成像，将新鲜切除腘窝淋巴结，脾，肺，或颅骨骨样品未​​切割到35 mm数0盖玻片的培养皿，在50〜100μLDMEM，10％FBS的20毫​​米肝素钠在37℃并利用双光子钛萨激发（860 nm）的结合激发焦（458纳米，488纳米，514纳米，561纳米，594纳米）和徕卡共振扫描仪（8000赫兹/秒），以Z-堆栈（〜为80μm），在20秒的间隔长达1小时。 <ol><li>对于同时三种颜色2P成像采用TI萨调整在860 nm处同时与OPO激光（调谐在1,130 nm波长tdTomato;或1140纳米mCherry）（ 电影2）。 </li></ol></li><li>三维体积和四维时间序列的重建与分析<ol><li>使用了Imaris 64软件是版本7.6，或替代的软件包来转换平铺电池堆的原始图像到容积重建（3D）的骨髓和不同的组织，并将其输出为3D旋转电影内的荧光细胞的数据，如前面所描述的3,4 （ 电影1）一步一步的例子： <ol><li>在了Imaris，打开Z堆栈的图像;为了显示3D体积可视化选择“超越”。 </li><li>使用“浏览”，打开音量360°。 </li><li>选择“动画”制作动画;使用关键帧添加所选的意见，变焦，音量的旋转;预览影片，根据需要进行编辑和保存在文件中所需的影片格式（如：AVI，MOV）。 </li></ol></li><li>对于三维细胞和克隆频率，位置，大小和分布进行定量评估，进一步的处理使用了Imaris XT模块的Z堆栈的集成进行距离MEAS MATLAB应用程序urements（使用距离变换算法）和簇分析，如先前所描述的3,4。变换的图像栈的序列随时间成体积渲染四维（4D）电影了Imaris 64的软件，并使用自回归运动算法在三维空间中使用的点和面分析为细胞运动的半自动跟踪。 </li><li>如下进行细胞的多谱分析：提取多色加入所有5个通道通过了Imaris XT通道运算到一个新的通道标记点;确定所有点（细胞）中的每一个的5分量信道的平均强度的荧光。在导出的Excel软件中的值绘制的图表显示了独特的“COLORPRINT”，即每个FP个体细胞（图2E图）的相对％。纠正组织漂移的数据集，并计算细胞的速度和位移随时间推移，轨迹长度，路径和距离;在Excel中SOF出口值tware绘制图形。组装与Adobe Photoshop的CSS 5软件的复合数字。 </li></ol></li></ol>

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作者宣称没有竞争的经济利益。

在这里，我们描述了一个功能强大的方法，最近发明了克隆细胞跟踪的细节，用相结合的组合与5FP编码LEGO载体和成像串联共聚焦和双光子显微镜标记来实现体积和动态成像活组织中产生大量的多样性。这延长了使用的FP为主色调，通过记录共焦光谱身份5“独特”的8位通道标识。因此蔚蓝，绿色荧光蛋白，金星，tdTomato，mCherry的每一个细胞内的相对比例创造了独特的“COLORPRINT”鉴定克隆后代。由5...

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

血细胞，称为造血的生产，是由一个小群体的造血干细胞和祖细胞（HSPCs）的维护。这些细胞位于骨髓（BM）中的一个复杂的微环境的生态位选自成骨细胞，基质细胞，脂肪组织，和血管结构之内，所有牵连的自我更新和分化1,2的控制。由于完整的BM向来人迹罕至的直接观察，HSPC及其微环境之间的相互作用仍然在体内主要是未鉴定。以前，我们建立了一个方法，用共聚焦荧光和反射镜3形象化完整的BM的3D架构。我们的特点扩大了BM绿色荧光蛋白标记的HSPCs，但只使用一个单一的FP排除再生分析，在克隆的水平。就在最近，我们又利用慢病毒基因本体论（LEGO）的载体组成型表达FLuorescent蛋白（fps）的有效标记细胞4,5。共转导HSPC与3或5向量生成的组合色彩多样的调色板，允许多个独立的HSPC克隆跟踪。 标记HSPC结合允许跟踪各个HSPC归巢和植入辐射小鼠的骨髓新的成像技术。 LEGO载体用于3或5个不同的帧率（从蔚蓝，绿色荧光蛋白，金星，tdTomato和mCherry）标记HSPC，并按照他们的植入时间的推移，在骨髓，激光共聚焦和双光子显微镜，让清晰的骨骼和其他可视化矩阵结构和HSPC克隆到其微环境的部件的关系。 HSPC纯化来自C57BL / 6小鼠骨髓谱系阴性细胞，转导的LEGO矢量，并通过尾静脉注射回输入髓烧蚀同类系小鼠。通过监测胸骨骨髓组织中大量的可视化，我们在年初发生的骨内植入骨髓再生。细胞簇与不同颜色的光谱出现最初在靠近骨和进展集中随着时间的推移。有趣的是，经过3个多星期的骨髓由宏观克隆簇，提示造血个别HSPCs衍生的连续传播​​的精髓，而不是HSPCs通过血液广泛传播。有较少的颜色多样性在晚的时间点，这表明难以传感长期再植细胞与多个矢量。 此外，HSPCs在脾脏形成集群，器官还负责造血小鼠。 HPSC衍生的单个细胞可在胸腺中得到解决，淋巴结，脾，肝，肺，心脏，皮肤，骨骼肌，脂肪组织和肾脏以及。 3D图像可以定性和定量地进行评估，以欣赏细胞与组织的最小扰动的分布。最后，我们说明在四维实时动态研究ð结合共振扫描多光子显微镜和共聚焦时间推移成像的可行性。这种方法使频谱标记的细胞群体的非侵入性的高解析度，多层面的细胞命运追踪其完整的3D架构，提供组织再生和病理学研究的有力工具。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Production of viruses</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-Cer2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27338</td>
			<td>Expression of Cerulean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-G2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">25917</td>
			<td>Expression of eGFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-V2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27340</td>
			<td>Expression of Venus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-T2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27342</td>
			<td>Expression of tdTomato</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-C2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27339</td>
			<td>Expression of mCherry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Calciumm Phosphate Transfection Kit</td>
			<td>Sigma</td>
			<td>CAPHOS-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Tissue culture dish</td>
			<td style="width:493px;">BD Falcon</td>
			<td style="width:216px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">IMDM</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Fetal Bovine Serum Heat Inactivated</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pen Strep Glutamine</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Centrifuge Tubes</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">326823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SW28 Ultracentrifuge</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">L60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Millex Syringe Driven Filter Unite (0.22um)</td>
			<td style="width:493px;">Millipore</td>
			<td style="width:216px;">SLGS033SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse cell collection, purification, transduction and transplantation</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">ACK lysing buffer</td>
			<td style="width:493px;">Quality Biologicals Inc.</td>
			<td style="width:216px;">118-156-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Lineage Cell Depletion Kit (mouse)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LS columns + tubes</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pre-Separation Filters (30um)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:493px;">StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500mL)</td>
			<td>StemCell Technologies Inc&#160;</td>
			<td>9650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">murine IL-11 CF</td>
			<td>R &#38; D Systems Inc</td>
			<td>418-ML-025/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine SCF</td>
			<td style="width:493px;">RDI Division of Fitzgerald Industries Intl&#160;</td>
			<td>RDI-2503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine IL-3</td>
			<td>R &#38; D Systems Inc</td>
			<td>403-ML-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">FLT-3 Ligand</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-096-480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">12-well plates, Costar</td>
			<td style="width:493px;">Corning Inc.</td>
			<td style="width:216px;">3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Retronectin</td>
			<td style="width:493px;">Takara Bio Inc</td>
			<td style="width:216px;">T100A/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Protamine Sulfate</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">P-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Confocal and two-photon microscopy</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">DMEM</td>
			<td style="width:493px;">Lonza</td>
			<td style="width:216px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">1M Hepes</td>
			<td style="width:493px;">Cellgro, Mediatech Inc.</td>
			<td style="width:216px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-20-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-14-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td>
			<td>Thermo Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Vision II -TiSaph laser range 680-1080nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Compact OPO laser range 1030-1350nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCX-IRAPO-L 25x/0.95 NA water dipping objective (WD=2.5 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PLAPO-CS 20x/0.70 NA dry objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PL-IRAPO 40x/1.1NA water immersion objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Imaris software version 7.6</td>
			<td style="width:493px;">Bitplane</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

我们开发并验证了一种荧光标记的方法对造血干细胞和祖细胞（HSPCs）以高空间和时间分辨率使用小鼠骨髓移植实验模型体内造血研究的克隆跟踪。遗传组合使用的编码荧光蛋白（FPS）的慢病毒载体标记通过先进的显微成像使细胞命运的映射。矢量编码五种不同的FPS：蔚蓝，绿色荧光蛋白，金星，tdTomato和mCherry被用来同时转导HSPCs，创建色彩标记的细胞多样的调色板。使用共聚焦/双光子混合显微成像能够唯一地标记细胞的同时高分辨率评估和其与组织的结构部件结合的后代。体积分析在大面积显示，频谱编码HSPC来源的细胞，可以检测非侵入性地在各个完整的组织，包括骨髓（BM），对于时间移植后广泛周期。现场研究相结合的视频速率多光子激光扫描共聚焦时间推移成像的4D演示动态的细胞和克隆跟踪以定量的方式的可能性。

全安装3D共焦胸骨骨髓时程/ 2 - 光子显微镜的重建揭示了移植和扩展移植联合5FPS的具有显着特征的模式：克隆出现了明确界定，均匀标有颜色的调色板宽，初期进展随着时间的推移优先包含多一种颜色的细胞。共焦显微镜的设置和成像5fps的标记HSPC在胸骨骨髓代表性实例示于图2和视频1和2。 完整组织的整个贴三维共聚焦/双光子显微镜重?...

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

组合5的荧光蛋白的造血干细胞和祖细胞的标记允许在通过共聚焦和双光子显微镜体内克隆跟踪，再生期间提供见解骨髓造血体系结构。此方法允许频谱编码HSPCs来源的细胞在完整组织的时间移植后广泛周期的非侵入性的命运映射。

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

in-vivo-clonal-tracking-hematopoietic-stem-progenitor-cells-marked

Research

JoVE Journal

Biology

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

13.1K 次观看.  NHLBI/NIH. 组合5的荧光蛋白的造血干细胞和祖细胞的标记允许在通过共聚焦和双光子显微镜体内克隆跟踪，再生期间提供见解骨髓造血体系结构。此方法允许频谱编码HSPCs来源的细胞在完整组织的时间移植后广泛周期的非侵入性的命运映射。 

视频: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

Methods Article

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪